eNAMPT neutralization reduces preclinical ARDS severity via rectified NFkB and Akt/mTORC2 signaling

Despite encouraging preclinical data, therapies to reduce ARDS mortality remains a globally unmet need, including during the COVID-19 pandemic. We previously identified extracellular nicotinamide phosphoribosyltransferase (eNAMPT) as a novel damage-associated molecular pattern protein (DAMP) via TLR4 ligation which regulates inflammatory cascade activation. eNAMPT is tightly linked to human ARDS by biomarker and genotyping studies in ARDS subjects. We now hypothesize that an eNAMPT-neutralizing mAb will significantly reduce the severity of ARDS lung inflammatory lung injury in diverse preclinical rat and porcine models. Sprague Dawley rats received eNAMPT mAb intravenously following exposure to intratracheal lipopolysaccharide (LPS) or to a traumatic blast (125 kPa) but prior to initiation of ventilator-induced lung injury (VILI) (4 h). Yucatan minipigs received intravenous eNAMPT mAb 2 h after initiation of septic shock and VILI (12 h). Each rat/porcine ARDS/VILI model was strongly associated with evidence of severe inflammatory lung injury with NFkB pathway activation and marked dysregulation of the Akt/mTORC2 signaling pathway. eNAMPT neutralization dramatically reduced inflammatory indices and the severity of lung injury in each rat/porcine ARDS/VILI model (~ 50% reduction) including reduction in serum lactate, and plasma levels of eNAMPT, IL-6, TNFα and Ang-2. The eNAMPT mAb further rectified NFkB pathway activation and preserved the Akt/mTORC2 signaling pathway. These results strongly support targeting the eNAMPT/TLR4 inflammatory pathway as a potential ARDS strategy to reduce inflammatory lung injury and ARDS mortality.

vitro and in vivo screening of murine mAb-derived humanized variants as we have previously described 9 .
Endothelial cell (EC) siRNA transfection. Human pulmonary artery endothelial cells (EC) were cultured in essential growth medium (EGM-2) containing 10% fetal bovine serum (Lonza, Walkersville, MD). Cells were placed in incubator at 37°C, 5% CO 2 and 95% humidity to achieve contact-inhibited monolayers as we previously described 9 . EC were transfected with siRNA (100 nM, GE Dharmacon, Lafayette, CO) specific for UCHL1 or non-specific scrambled sequence using transfection reagent siPORT Amine (Ambion, Austin, TX) in serum-free conditions according to the manufacturer's protocol. The medium was changed to EGM-2 containing 2% fetal bovine serum after 24 hrs of transfection and protein silencing was checked after 72 hrs of transfection. Silenced cells were utilized for western blotting studies described below.
Animals utilized. Sprague Dawley male rats (300-350 g) were purchased from Charles River (Wilmington, MA). For porcine experiments, Yucatan male minipigs (17-20 kg) were purchased from S&S Farms (Ramona, California). All rats and minipigs were housed under standard conditions (12 h light-dark cycle, 25-27 C⁰, 40% humidity). Rats were kept in autoclaved microisolator cages or pens with free access to food and water throughout the duration of the experiments. All animal care procedures and experiments were approved by the Institutional Animal Care and Use Committee (University of Arizona).
LPS "one-hit" preclinical ARDS rat model. Sprague Dawley rats were anesthetized with a mixture of ketamine (100 mg/kg) and xylazine (5 mg/kg) (intraperitoneal injection). Animals were intratracheally intubated with a 16-G angiocatheter which was used for LPS instillation (E. Coli 0127: B8, 1 mg/kg) and harvested 18 h later as we reported 17 . LPS/VILI "two-hit" preclinical ARDS rat model. Sprague Dawley male rats were anesthetized and LPS (0.1 mg/kg) intratracheally delivered as described in the "one-hit " LPS model. After 18 h, rats were re-anesthetized, intratracheally reintubated and connected to mechanical ventilation (Advanced Ventilator System for Rodents, SAR-1000, CWE Incorporated, Ardmore, PA) as previously described 17 18 . Supplemental ketamine and xylazine was provided to ensure adequate anesthetic depth during mechanical ventilation. Rats were ventilated for 4h with room air, tidal volume (Vt) 20 ml/kg, respiratory rate (RR) 70 breaths/min, and positive-end expiratory pressure (PEEP) 0 cm H2O. Spontaneously breathing (SB) control animals received intratracheal PBS instead of LPS and following 18 h period, allowed to breathe spontaneously on room air for 4 h before harvesting.
Blast trauma/VILI "two-hit" preclinical ARDS rat model. Studies utilizing the rat model of blast trauma/VILI were conducted at the US Army Institute of Surgical Research (Fort Sam Houston, TX). Sprague Dawley male rats (10-12 weeks old, 350~475g) were anesthetized with a mixture of intraperitoneal ketamine (100 mg/kg) and xylazine (5 mg/kg) and isoflurane (1-3%) used to maintain anesthesia depth during mechanical ventilation. Carotid artery and jugular vein catheters were placed in anesthetized animals which were then placed in prone position with the head facing the blast front, and secured on a holder consisting of a flat plastic mesh suspended between two stainless steel rods, which run horizontally alongside the animal. A second plastic mesh was placed over the top of the rat to further secure the rat. The holder was attached to a swing 4 mechanism to allow the rat to partially recoil against the blast. Animals were then exposed to a blast overpressure [BOP = 150.4 kPa, duration of positive phase overpressure (t+) = 3.4 ms) using a compressed air-driven shock tube (Applied Research Associates, Littleton, CO). Rats were retrieved after blast exposure, observed for 30 min, then placed on mechanical ventilation for 4 hs (RR of 70 breaths/min, tidal volume (Vt) 10 ml/kg, 0 PEEP) using the VentElite small animal ventilator (Harvard Apparatus).
Septic shock/VILI "two-hit" preclinical ARDS porcine model. Male Yucatan minipigs were anesthetized with isoflurane to induce the anesthesia, followed by IV anesthesia (TIVA) with propofol (5-15mg/kg/h), ketamine (2-6mg/kg/h), and midazolam (0.25-0.75mg/kg/h) to maintain anesthesia during the 12h study. Two venous catheters were placed and secured into the auricular and dorsal pedal veins. The femoral artery was cannulated for mean arterial pressure (MAP) monitoring and hourly arterial blood sampling for arterial blood gases (ABG) (pH, pCO2, pO2, BEecf, HCO3, SpO2, and lactate), chemistries and electrolytes (I-Stat, CG4+, CHEM8+ cartridges). Animals were intratracheally intubated and connected to a Galileo mechanical ventilator (Hamilton Medical) on volume assist-control mode. Animals received continuous clinical monitoring for the entire 12-hr ICU duration: heart rate (HR), respiratory rate (RR), electrocardiogram, oxygen saturation (SpO2), core temperature, end tidal carbon dioxide (ETCO2) (BM5 VET ICU monitoring system). At the onset of the experiment, pigs received IV LPS (25ug/kg, E. Coli 0127-B8, Sigma Co) infused over a 2 hrs period while pigs received 100% O2 and were ventilated with a tidal volume (Vt) of 13 ml/kg, RR of 15 breaths/min, and PEEP of 5 cm H2O. After the 2 hrs LPS infusion, the Vt was increased to 20ml/kg, PEEP of 5 cm H2O and the RR adjusted to maintain ABG values within normal range. Bronchoalveolar lavage (BAL) was performed using the disposable flexible bronchoscopy (Ambuâ) at the beginning (baseline sample) and at 12 hrs study end. BAL samples were used to obtain BAL total and PMN cell counts as well as BAL protein levels.
Delivery of the eNAMPT-neutralizing antibodies in rat and porcine ARDS/VILI preclinical models. Intravenous eNAMPT-neutralizing pAb (4 mg/kg) or the eNAMPT mAb (0.4 mg/kg) was delivered concomitantly with LPS challenge in the "one-hit" LPS model, or with the "two-hit" LPS/VILI model. For the blast trauma/VILI "two-hit" rat model, specific groups of rats received intravenous eNAMPT pAb (4mg/kg) after blast exposure (0.5 h), prior to initiation of mechanical ventilation. For the porcine septic shock/VILI preclinical model, one group of pigs received either intravenous eNAMPT mAb (0.4 mg/kg) or PBS at the end of LPS infusion period, prior to high tidal volume ventilation exposure.

Bronchoalveolar lavage (BAL) analysis.
At the termination of each experiment, rats were euthanized by approved IACUC methods (exsanguination after anesthesia). BAL was performed with 4 ml of cold Hank's buffered saline solution (HBSS) (Invitrogen) delivered intratracheally to the left lung after placing a surgical suture on the right lung followed by slow recovery of the BAL fluid as we have previously described 9 17 19 . In the porcine "two hit" model, BAL fluid collection was performed at two time points: at the beginning of the study prior to LPS instillation (baseline sample), and at the endpoint of study (12h). Cold Hank's buffered saline (HBSS, 10 ml) was instilled via a disposable flexible bronchoscopy (Ambuâ). BAL fluid from both rat and porcine BALs were processed as we have described 9 17 19 with centrifugation (500g, 20 min, 4°C) and the pellets re-suspended in 200µL of cold HBSS. RBC Lysis Solution from Qiagen (1ml-5min) was 6 used to eliminate RBC in the lavage. Samples were recentrifuged (500g, 10 min, 4°C) then pellets were re-suspended in 200µL of cold HBSS for total cell counting, using an automated cell counter (TC20; Bio-Rad, Hercules, CA) and for differential cell count to detect the number of PMNs by using cytospin centrifuge machine from Thermo Scientific (600 rpm, 25min) and Diff-Quick staining kit. The BAL supernatant was re-centrifuged (16,5000g, 10 min, 4°C), and the supernatant was collected for total protein measurements (Pierce BCA Protein Assay Kit, Thermo Scientific).
BAL supernatant was stored at -80°C for further analysis.

Quantitative lung histology and immunohistochemistry (IHC) analyses. To assess alterations
in the lung tissue morphology, lungs collected from sacrificed rats and pigs were fixed in 10% neutral buffered formalin for a minimum of 48 h, embedded in paraffin, sectioned, mounted onto slides, and stained with hematoxylin-eosin (H & E). Routine H&E slides were prepared using Richard-Allan hematoxylin, clarifier, bluing reagent and eosin as we have previously described 9 .
The avidin-biotin-peroxidase method was utilized for IHC staining to visualize NAMPT expression in lung tissues (5 micron sections) (Bethyl Laboratories, Montgomery TX) or a rabbit IgG control (matched protein concentration, 1ug/ml, Vector Labs, Burlingame CA).
Deparaffinized and rehydrated slides were ringed with an ImmunoPen rinsed in TBS, blocked for endogenous peroxidase using freshly prepared 0.5% hydrogen peroxide, 20 min, washed, protein block (Vector Labs) for 1 h, followed by avidin D and biotin block (Vector Labs), all at room temperature. Slides were incubated in primary or IgG isotype control, overnight at 4 o C with humidity. After washing, biotinylated secondary was applied for 1 h, washed and incubated with avidin-biotin complex (Vector Labs) 40 min, all at room temperature. The protein was visualized using DAB plus nickel (Vector Labs), 4 min, rinsed in tap water and counterstained with Mayers hematoxylin (Newcomer Supply, Middleton, WI) 30 sec, washed in water, bluing reagent (Richard-Allan Scientific, San Diego CA) 10 sec, washed in water, dehydrated, cleared and coverslipped with DPX.
Formalin-fixed paraffin embedded tissue sections were baked at 65 o C overnight, washed in xylene 3 times, 7 minutes each, to remove paraffin, followed by passing through 100%, 75%, 50% isopropanol, and ddH20 for rehydration. Antigen retrieval was performed using a sodium Specimens were imaged using Zeiss Axiovert Photomicroscope using a 10X objective (NA 0.4). Histological images from each group captured with light microscopy (Olympus digital camera) at 10x magnification, were randomly selected for quantification of H&E and NAMPT staining using ImageJ software 9 (different sections of each slide). For H&E image analysis, the percentage of area selected for measurement with all images processed and stored for statistical analysis. For NAMPT staining image analysis, color segmentation plugin was utilized with POINTCROSS tool applied to each NAMPT staining image with a total of 3 color clusters. The 8 area percentage of each color cluster was recorded and saved for statistical analysis as we have reported 9 .

Plasma and BAL biomarker measurements.
A meso-scale ELISA platform (Meso Scale Diagnostics, Rockville, MD) was utilized as we previously described 9 for measurements of plasma levels of eNAMPT, IL-6 and TNFa in rats and eNAMPT, IL-6, IL-1RA and angiopoetin-2 in pigs.
Following incubation 5 min at 90°C in loading buffer, aliquots containing equal amounts of protein (25-30 ug) were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Subsequently, proteins were transferred to PVDF membranes and probed with specific primary antibodies by horseradish peroxidase-conjugated secondary antibodies. Proteins were visualized using an ECL system (Pierce West Pico cat #34580) and ChemiDoc MP imaging system (Bio-Rad). Densitometric analysis was performed using Bio-Rad Image Lab 6.01 software by normalizing the levels of proteins to b-actin expression. The levels of phosphor-proteins were quantified by normalizing the levels to their respective total proteins 9 .

Reactive oxygen species (ROS) measurements in porcine lung tissue:
The generation of reactive oxygen species was measured using electron paramagnetic resonance spectroscopy (EPR).
Briefly, ~50-100 mg of lung tissue was incubated with 20 mM Krebs-HEPES buffer (pH 7.4) containing 200 μM CMH for 30 min. Following treatment, buffer was collected, and changes in CMH oxidation were measured for 15 min using the e-scan Multipurpose Bench-top EPR system (Noxygen Science and Transfer Diagnostics GmbH). CMH signal is represented as nM/min/mg of lung tissue, and all treatment groups were normalized to controls. Statistical analysis. Continuous data were compared using nonparametric methods and categorical data by chi square test. Where applicable, standard one-way ANOVA was used and groups were compared using the Newman-Keuls test. Differences between groups were considered statistically significant when p values were less than 0.05 (p <0.05). Two-way ANOVA was used to compare the means of data from two or more different experimental groups. If significant differences were present by ANOVA (p <0.05), a least significant differences (LSD) test was